What Is the Purpose of Polyacrylamide Gel Electrophoresis (PAGE)?

An Overview

Polyacrylamide gel electrophoresis, or PAGE, is a powerful tool used by scientists to separate and analyse proteins, DNA, and RNA. This technique is widely used in various fields, including biochemistry, molecular biology, genetics, and proteomics, to study the properties of macromolecules and their interactions.

Polyacrylamide Gel Electrophoresis with 15-Lane Blue Loading Buffer

Given below is the purpose of polyacrylamide gel electrophoresis, its history, and how it works. We will also elucidate the different types of PAGE and their applications, as well as the advantages and limitations of this technique.

History of Polyacrylamide Gel Electrophoresis

The development of polyacrylamide gel electrophoresis can be traced back to the 1950s, when it was first used to separate proteins by Tiselius and Kabat. However, it wasn't until the 1960s that polyacrylamide gel electrophoresis became a widely used technique in biochemistry and molecular biology.

Polyacrylamide gel electrophoresis was developed as an improvement over earlier electrophoresis techniques, such as paper and cellulose acetate electrophoresis, which had limited resolution and sensitivity.

The use of polyacrylamide gel as a matrix for electrophoresis allowed for the separation of molecules based on their size, shape, and charge, providing a more precise and accurate way to analyse proteins, DNA, and RNA.

How Polyacrylamide Gel Electrophoresis Works

Polyacrylamide gel electrophoresis works by separating macromolecules based on their size and charge. In this technique, a sample containing the molecules of interest is loaded onto a gel matrix made of polyacrylamide, a synthetic polymer that forms a porous network when polymerized.

The gel matrix is placed in a buffer solution that provides an electric field, causing the molecules in the sample to move through the gel matrix based on their charge. The gel matrix acts as a molecular sieve, separating the molecules based on their size, with smaller molecules moving more quickly through the gel than larger molecules.

Once the molecules have been separated, they can be visualized and analysed using various staining and detection methods, such as Coomassie Blue staining, silver staining, or Western blotting.

Types of Polyacrylamide Gel Electrophoresis

There are two main types of polyacrylamide gel electrophoresis: SDS-PAGE and Native-PAGE.

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

SDS-PAGE is a widely used technique that separates proteins based on their size alone. In this technique, a detergent called SDS is used to denature the proteins, causing them to unfold and assume a linear shape.

The SDS molecules also impart a negative charge to the proteins, allowing them to move through the gel matrix based solely on their size. DS-PAGE is commonly used for protein purification, quantification, and analysis. It is also used in the Western blotting technique to detect specific proteins in a sample.

Loading Sample in to SDS-PAGE

Native-PAGE

Native-PAGE is a technique that separates proteins based on their size and charge. Unlike SDS-PAGE, Native-PAGE does not denature the proteins, allowing them to retain their native conformation and charge.

Native-PAGE is commonly used to analyse protein complexes and their interactions, as well as to study the structure and function of enzymes.

Advantages of Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis has several advantages over other separation techniques −

• High Resolution: Polyacrylamide gel electrophoresis can separate molecules with high resolution, providing a more accurate analysis of complex mixtures of macromolecules.

• Quantification: Polyacrylamide gel electrophoresis can be used to quantify the amount of protein or nucleic acid in a sample.

• Sensitivity: Polyacrylamide gel electrophoresis can detect proteins and nucleic acids at very low concentrations, making it a useful technique for analyzing samples with limited amounts of material.

• Versatility: Polyacrylamide gel electrophoresis can be used to separate and analyse a wide range of macromolecules, including proteins, DNA, and RNA.

Limitations of Polyacrylamide Gel Electrophoresis

Despite its advantages, polyacrylamide gel electrophoresis also has some limitations −

• Time-consuming: Polyacrylamide gel electrophoresis can be a time-consuming technique, particularly when analysing complex mixtures of macromolecules.

• Limited sample capacity: Polyacrylamide gels have a limited capacity for sample loading, making it difficult to analyse large amounts of material.

• Non-specific detection: Polyacrylamide gel electrophoresis does not provide specific information about the identity of the macromolecules being separated. Additional detection methods, such as Western blotting, are often required to identify specific proteins.

Applications of Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis has a wide range of applications in various fields, including −

• Protein Analysis: Polyacrylamide gel electrophoresis is commonly used to analyse proteins, including their size, abundance, and interactions.

• Nucleic Acid Analysis: Polyacrylamide gel electrophoresis can be used to separate and analyze DNA and RNA molecules, including their size and purity.

• Enzyme analysis: Polyacrylamide gel electrophoresis can be used to analyze the activity and structure of enzymes.

• Diagnostic testing: Polyacrylamide gel electrophoresis can be used for diagnostic testing, such as detecting abnormal proteins or mutations in DNA.

• Quality control: Polyacrylamide gel electrophoresis can be used for quality control of pharmaceutical products, such as vaccines and biologics.

Conclusion

Polyacrylamide gel electrophoresis is a powerful technique used to separate and analyse proteins, DNA, and RNA. This technique has been used for over 50 years and continues to be an essential tool in biochemistry, molecular biology, genetics, and proteomics.

Polyacrylamide gel electrophoresis provides high-resolution separation of macromolecules and can be used for quantification and sensitivity analysis. However, it also has some limitations, including being time-consuming and having limited sample capacity.

Overall, polyacrylamide gel electrophoresis remains a versatile and widely used technique that continues to advance our understanding of macromolecules and their interactions.